Self-assembling icosahedral capsids are found in about half of known virus families. Yet, the mechanisms of assembly remain a black box, even though assembly is fundamental to biology. For example, assembly is critical to, cellular localization, nucleic acid packaging, and new classes of assembly-directed antivirals (sometimes described as assembly inhibitors though they actually promote mis-assembly). To date, almost all observations of assembly have depended on ensembles of structurally ill-defined complexes. Similarly, almost all models of assembly have depended on ad hoc assumptions regarding the state of subunits in solution, the state of subunits in incomplete capsids, and the list of intermediates participating in assembly. We propose a detailed analysis of the assembly of Hepatitis B Virus (HBV). In vitro experiments, involving mutants and combinations of mutants will be validated by comparison to assembly in cell culture. Based on modeling studies we hypothesize that assembly of the 120-dimer T=4 HBV capsid will involve a relatively small number of intermediates. If this is the case it has immediate implications for the organization of viral RNA, which in vivo is packaged during the assembly process. In this proposal we will characterize HBV capsid self-assembly using biochemical techniques and electron microscopy (Zlotnick, Wang labs), time resolved SAXS (Raviv Lab), and single particle observations by charge detection mass spectrometry (Jarrold Lab). Preliminary data show the feasibility of experiments and indicate the presence of some favored intermediates. These results will contribute to the development of mathematical models that faithfully replicate assembly and can help identify salient points for interfering with normal assembly. HBV is one the smallest human pathogens (3200 bp DNA genome) and one of the most widespread. About 350 million people have chronic HBV leading to about 600,000 deaths each year. Current therapeutics suppress but do not cure infection. Several pharmaceutical companies are investigating HBV capsid protein as an antiviral target with potential to achieve a functional cure